The present invention relates to the fields of pharmacology and drug discovery. More particularly, the invention relates to novel peptide compositions that can bind and activate the human erythropoietin receptor, and to methods of making small molecule agonists of the erythropoietin receptor using such peptide compositions as design templates.
Drug discovery traditionally has relied upon high-throughput screening of large numbers of chemical compounds to identify novel drug leads. More recently, combinatorial libraries constructed by chemical or biological means have greatly expanded the number of compounds available for screening. Biological libraries, such as phage displayed peptide libraries, of random directed semi-random sequences represent particularly rich sources of molecular diversity and advantageously possess the ability to self-replicate. With a self-replicating system, the search for high affinity leads is not limited to members that happen to be present in the initial library. As discussed more fully below, desired characteristics of initial sequences can be greatly improved by employing successive rounds of mutagenesis, affinity selection, and amplification. These approaches recently have been used to discover small peptides capable of binding several cytokine receptors.
Erythropoietin (EPO) is a cytokine that stimulates the formation of red blood cells by inducing the growth and differentiation of progenitor cells. The recombinant version of human EPO is a valuable therapeutic agent useful for treating anemia that is associated with several pathological conditions, including chronic renal failure, malignancy or the effects of chemotherapy, HIV and rheumatoid arthritis. When used therapeutically, EPO must be administered either by intravenous or subcutaneous injection. The fact that EPO is a relatively large glycoprotein adversely impacts the cost of manufacture, the pharmacological properties of molecule, and the mode of delivery of this therapeutic agent.
The erythropoietin receptor (EPOR) belongs to the cytokine receptor superfamily whose members share common structural features including an extracellular ligand binding domain, a single transmembrane-spanning region, and. an intracellular cytoplasmic domain. The extracellular domain (ECD) is sufficient to mediate receptor-ligand binding. It is therefore possible through recombinant DNA techniques to synthesize DNA encoding the ECD as a fusion with secreted proteins to produce reagents useful for identifying receptor binding molecules, for example by screening a phage display library.
Phage display libraries expressing fusions of random or semi-random peptides and bacteriophage coat proteins represent convenient versions of combinatorial libraries that can be screened to identify receptor ligands. Upon infection and assembly of phage particles, the random polypeptides are outwardly disposed for interaction with antibodies or other receptor probes. Since the phage particles contain the nucleic acid that encodes the fusion protein, the genetic information which identifies the sequence of the fusion protein is physically linked to the fusion protein. Construction and screening of such phage expression libraries are well known and have been described previously, such as, for example, in Sawyer et al., Protein Engineering 4:947-953 (1991); Akamatsu et al., J. Immunol. 151:4651-59 (1993), Smith et al., Methods in Enzymol. 217:228-257 (1993); Clackson et al., Trends Biotechnol. 12:173-184 (1994), and U.S. Pat. No. 5,427,908 to Dower et al.
In one example of a screening procedure, a soluble form of the EPOR was used to probe a phage display library to identify candidate peptides having EPO-like properties. Wrighton et al., in Science 273:458 (1996) described the use of a fusion protein comprising the EPOR extracellular domain and human placental alkaline phosphatase in a library screening protocol. The library used for this purpose displayed cyclic 8-residue peptides as fusions with the pVIII coat protein of a filamentous phage. Peptides having higher affinity for the EPOR were subsequently isolated from mutagenesis libraries that displayed pIII protein fusions. This approach led to the identification of several peptides that stimulated erythropoiesis in mice. These agonists were disulfide-bonded cyclic peptides having the minimum consensus sequence YXCXXGPXTWXCXP (SEQ ID NO:1), where X is a position that can be occupied by any of several amino acids.
Despite this progress toward identifying EPOR ligands, there remains a need to identify ligands exhibiting superior receptor-binding properties as well as ligands that bind to the receptor using previously unknown contact sites.
According to one aspect of the invention, there is disclosed an isolated polypeptide capable of binding to a human erythropoietin receptor, having the formula:
Xn-C-X1-X2-G-W-V-G-X3-C-X4-X5-W-XC
wherein Xn is an amino-terminal peptide of from 2 to 4 natural xcex1-amino acids in length; XC is a carboxy-terminal dipeptide; and X1, X2, X3, X4 and X5 are independently selected from the group consisting of natural a-amino acids.
In preferred embodiments of the isolated polypeptide, the amino-terminal Xn is Xn1-Xn2-Xn3-Xn4, wherein Xn1 is selected from the group consisting of neutral and polar, neutral and hydrophobic, and acidic natural a-amino acids, or optionally Xn1 is absent; Xn2 is selected from the group consisting of neutral and polar, neutral and hydrophobic, and basic natural a-amino acids, or optionally Xn2 is absent if Xn1 is absent; Xn3 is selected from the group consisting of natural xcex1-amino acids; and Xn4 is selected from the group consisting of neutral and polar, neutral and hydrophobic, and acidic natural xcex1-amino acids. More preferably, Xn1 is E, G, N, S, D, Q, L, Y or A; Xn2 is F, V, A, K, R, G, S, I or L; Xn3 is H, Q, E, G, D, A, or V; and Xn4 is E, G, V, A, P, D, T or M. In other preferred embodiments, Xn1 is absent; Xn2 is F, V, A, K, R, G, S, I or L; Xn3 is H, Q, E, G, D, A, or V; and Xn4 is E, G, V, A, P, D, T or M. In one embodiment Xn1 is an acidic amino acid. In another embodiment, Xn2 is a branched-chain amino acid or K. In yet another embodiment, Xn3 is an acidic amino acid or V. In one embodiment, Xn4 is V or G. In preferred embodiments, X1 is a neutral and hydrophobic, neutral and polar, or basic amino acid; more preferably X1 is R, I, G, Q, L, T or S; and most preferably, is R or I. In preferred embodiments, X2 is a neutral and hydrophobic, neutral and polar, or basic amino acid; more preferably, X2 is R, P, W, G, L or N; and most preferably is R. In preferred embodiments, X3 is a neutral and polar, or basic amino acid; and more preferably X3 is H, Q or N. In preferred embodiments, X4 is a neutral and polar, basic, or acidic amino acid; more preferably X4 is Q, N, S, K or E; and most preferably is K or N. In preferred embodiments, X5 is a neutral and polar, neutral and hydrophobic, or acidic amino acid; more preferably is V, A, Y, D or E; and most preferably is D or E. In preferred embodiments, the carboxy-terminal Xc is Xc1-Xc2 and wherein Xc1 is a neutral and polar, or neutral and hydrophobic amino acid, and Xc2 is a neutral and polar, neutral and hydrophobic, or basic amino acid. In preferred embodiments, Xc1 is L, I, P, F, M, Q or G; and more preferably is L or Q. In preferred embodiments, Xc2 is M, W, T, S, G, N or R; and, more preferably, Xc2 is G or R.
Other aspects of the invention are isolated polypeptides having the amino acid sequences of SEQ ID NO:81 and SEQ ID NO:82.
According to another aspect of the invention, there is disclosed a method of activating a human erythropoietin receptor, comprising the steps of contacting a cell having an erythropoietin receptor on it surface with a peptide mimetic of erythropoietin, wherein the peptide mimetic is a compound having the general formula
Xn-C-X1-X2-G-W-V-G-X3-C-X4-X5-W-XC
wherein Xn is an amino-terminal peptide of from 2 to 4 natural xcex1-amino acids in length; XC is a carboxy-terminal dipeptide; and X1, X2, X3, X4 and X5 are independently selected from the group consisting of natural xcex1-amino acids; and allowing the peptide mimetic to bind to the erythropoietin receptor, thereby initiating activation of the erythropoietin receptor.
Another aspect of the invention that is disclosed is a method of inhibiting binding of erythropoietin to an erythropoietin receptor, comprising the steps of providing a peptide mimetic having the general formula
Xn-C-X1-X2-G-W-V-G-X3-C-X4-X5-W-XC
wherein Xn is an amino-terminal peptide of from 2 to 4 natural xcex1-amino acids in length; XC is a carboxy-terminal dipeptide; and X1, X2, X3, X4 and X5 are independently selected from the group consisting of natural xcex1-amino acids, in sufficient quantity to compete with erythropoietin for binding to an erythropoietin receptor; and allowing the peptide mimetic to interact with the erythropoietin receptor, thereby inhibiting binding of erythropoietin to the erythropoietin receptor. In a preferred embodiment, the erythropoietin receptor is derived from a human.
Another aspect of the invention is a method of discovering drugs that mimic erythropoietin, comprising the steps of constructing a phage display library in which a fusion protein comprising a peptide consisting of a random sequence of amino acids and a phage protein; screening the phage display library for at least one clone that binds to a human erythropoietin receptor probe; isolating the clone that bind to a human erythropoietin receptor probe; determining a nucleic acid sequence from the clone that codes for the peptide contained within the fusion protein; constructing an evolved phage display library by mutagenesis in vitro of the nucleic acid sequence that codes for the peptide contained within the fusion protein; screening the evolved phage display library for clones that bind to a human erythropoietin receptor probe; isolating the clones from the evolved phage display library; determining nucleic acid sequences from the clones, wherein each nucleic acid codes for a peptide contained within the clone""s fusion protein; comparing the nucleic acid sequences to identify a consensus amino acid sequence; and synthesizing a compound that mimics the consensus amino acid sequence. In a preferred embodiment, the synthesizing step includes synthesizing a compound that is an organic compound, preferably a peptide. More preferably, the synthesized peptide is a cyclic peptide.